Erythropoietin (EPO) is a glycoprotein hormone involved in the growth and maturation of erythroid progenitor cells into erythrocytes. EPO is produced by the liver during fetal life and by the kidney of adults and stimulates the production of red blood cells from erythroid precursors. Decreased production of EPO, which commonly occurs in adults as a result of renal failure, leads to anemia. EPO has been produced by genetic engineering techniques involving expression and secretion of the protein from a host cell transfected with the gene encoding erythropoietin. Administration of recombinant EPO has been effective in the treatment of anemia. For example, Eschbach et al. (N. Engl J Med 316, 73 (1987)) describe the use of EPO to correct anemia resulting from chronic renal failure.
The purification of human urinary EPO was described by Miyake et al. (J. Biol. Chem. 252, 5558 (1977)). The identification, cloning, and expression of genes encoding erythropoietin is described in U.S. Pat. No. 4,703,008 to Lin. A description of a method for purification of recombinant EPO from cell medium is included in U.S. Pat. No. 4,667,016 to Lai et al.
Little is known about the mechanism by which EPO stimulates erythropoiesis. While it is clear that EPO activates cells to grow and/or differentiate by binding to specific cell surface receptors, the specific mechanism of activation as well as the structure of the receptor and any associated protein(s) is not completely understood. The erythropoietin receptor (EPO-R) is thought to exist as a multimeric complex. Sedimentation studies suggested its molecular weight is 330.+-.48 kDa (Mayeux et al. Eur. J. Biochem. 194, 271 (1990)). Crosslinking studies indicated that the receptor complex consists of at least two distinct polypeptides, a 66-72 kDa species, and 85 and 100 kDa species (Mayeux et al. J. Biol. Chem. 266, 23380 (1991)); McCaffery et al. J. Biol. Chem. 264, 10507 (1991)). A distinct 95 kDa protein was also detected by immunoprecipitation of EPO receptor (Miura & Ihle Blood 81, 1739 (1993)). Another crosslinking study revealed three EPO containing complexes of 110, 130 and 145 kDa. The 110 and 145 kDa complexes contained EPO receptor since they could be immunoprecipitated with antibodies raised against the receptor (Miura & Ihle, supra). Expression of a carboxy-terminal truncated EPO receptor resulted in detection of the 110 kDa complex but not the 145 kDa complex. This suggests that the higher molecular weight complex contains polypeptides present in the 110 kDa complex and an additional 35 kDa protein.
Further insight into the structure and function of the EPO receptor complex was obtained upon cloning and expression of the mouse and human EPO receptors (D'Andrea et al. Cell 57, 277 (1989); Jones et al. Blood 76, 31 (1990); Winkelmann et al. Blood 76, 24 (1990); PCT Application No. WO90/08822; U.S. Pat. No. 5,278,065 to D'Andrea et al.) The full-length human EPO receptor is a 483 amino acid transmembrane protein with an approximately 224 amino acid extracellular domain and a 25 amino acid signal peptide. The human receptor shows about an 82% amino acid sequence homology with the mouse receptor. The cloned full length EPO receptor expressed in mammalian cells (66-72 KDa) has been shown to bind EPO with an affinity (100-300 nM) similar to that of the native receptor on erythroid progenitor cells. Thus this form is thought to contain the main EPO binding determinant and is referred to as the EPO receptor. The 85 and 100 KDa proteins observed as part of a cross-linked complex are distinct from the EPO receptor but must be in close proximity to EPO because EPO can be crosslinked to them. The 85 and 100 KDa proteins are related to each other and the 85 KDa protein may be a proteolytic cleavage product of the 100 KDa species (Sawyer J. Biol. Chem. 264, 13343 (1989)).
A soluble (truncated) form of the EPO receptor containing only the extracellular domain has been produced and found to bind EPO with an affinity of about 1 nM, or about 3 to 10-fold lower than the full-length receptor (Harris et al. J. Biol. Chem. 267, 15205 (1992); Yang & Jones Blood 82, 1713 (1993)). The reason for the reduced affinity as compared to the full length protein is not known. There is a possibility that other protein species may also be part of the EPOR complex and contribute to EPO binding thus increasing the affinity. In support of this possibility is the observation of Dong & Goldwasser (Exp. Hematol. 21, 483 (1993)) that fusion of a cell line with a low affinity EPO receptor with a CHO cell which does not bind EPO resulted in a hybrid cell line exhibiting high EPO binding affinity of the receptor for EPO. In addition, transfection of a full length EPOR into CHO cells resulted in a cell line with both high and low affinity receptors as measured by Scatchard analysis. Amplification of the EPOR copy number increased the low affinity but not high affinity binding. These results are consistent with the presence of a limited quantity of a protein present in CHO cells that converts the low affinity EPOR to high affinity.
Activation of the EPO receptor results in several biological effects. Three of the activities include stimulation of proliferation, stimulation of differentiation and inhibition of apoptosis (Liboi et al. Proc. Natl. Acad. Sci. USA 90, 11351 (1993); Koury Science 248, 378 (1990)). The signal transduction pathways resulting in stimulation of proliferation and stimulation of differentiation appear to be separable (Noguchi et al. Mol. Cell. Biol. 8, 2604 (1988); Patel et al. J. Biol. Chem. 267, 21300 (1992); Liboi et al. ibid). Some results suggest that an accessory protein may be necessary for mediating the differentiation signal (Chiba et al. Nature 362, 646 (1993); Chiba et al. Proc. Natl. Acad. Sci. USA 90, 11593 (1993)). However there is controversy regarding the role of accessory proteins in differentiation since a constitutively activated form of the receptor can stimulate both proliferation and differentiation (Pharr et al. Proc. Natl. Acad. Sci. USA 90, 938 (1993)).
Activation of the EPO receptor may be due to its dimerization. That is, EPO may act as a crosslinker between two EPO receptor molecules. There is evidence in support of this proposal. An arginine to cysteine mutation at position 129 of the murine EPO receptor results in constitutive activation of the receptor, presumably because of a disulfide bond formed between two receptors subunits (Yoshimura et al. Nature 348, 647 (1990)). In addition EPOR is found in multimeric complexes in cells (Miura & Ihle Arch. Biochem. Biophys. 306, 200 (1993)). However, isolation of a stable multimeric form of purified EPO soluble receptor has not been reported. In addition, dimerization of EPOR may be required, but not by itself be sufficient for complete activation of cells. For example, dimerization may result in a proliferative signal but not a differentiation signal. That is, accessory proteins may be required to send the differentiation signal.
The possible relationship between EPO receptor dimerization and activation may be exploited to identify compounds which are different from EPO but activate the receptor. For example, antibodies possess two identical binding sites for antigen. An anti-EPOR antibody can bind two EPOR molecules and could bring them into close proximity to each other to allow dimerization. In order to function in vivo, these antibodies must recognize the EPOR on surfaces of cells and bind in a way that allows activation of the signal transduction pathway. In addition, it is desirable that activation result in both proliferation and differentiation of erythroid progenitors. A similar approach to understand the activation of human growth hormone receptor (Fuh et al. Science 256, 1677 (1992)) and epidermal growth factor receptor (Schreiber et al. Proc. Natl. Acad. Sci. USA 78, 7535 (1981)) has been reported
It would be desirable to identify molecules which have the property of activating the EPO receptor and stimulating erythropoiesis. In order to do so, an understanding of the mechanism of EPO receptor activation and signal transduction is important. One approach to elucidating this mechanism may be to identify antibodies which recognize the EPO receptor so as to activate the receptor and stimulate erythropoiesis. Such antibodies are useful in therapeutic and diagnostic applications and would also be useful for probing EPO receptor function.
The following references describe antibodies which bind to the mouse or human EPO receptor:
D'Andrea et al. in The Biology of Hemtaopoiesis, Wiley-Liss, Inc. (1990) pp. 153-159, generated polyclonal anti-peptide antibodies against an amino-terminal and a carboxy-terminal peptide of murine EPO receptor. The antibodies were shown to react with mouse EPO receptor in a Western blot.
Bailey et al. Exp. Hematol. 21, 1535-1543 (1993) generated polyclonal anti-peptide antibodies against synthetic peptides homologous to the extraceullular and cytoplasmic domains of the mouse EPO receptor. Receptor activation by these antibodies, as measured by 3H thymidine uptake into spleen cells from phenylhydrazine treated mice, was not detected .
Baynes et al. Blood 82, 2088-2095 (1993) generated a polyclonal antibody to an amino-terminal peptide in the human EPO receptor. The antibody was shown to react with a soluble form of the receptor present in human serum.
D'Andrea et al. Blood 82, 46-52 (1993) generated monoclonal antibodies to human EPO receptor. The antibodies bind to Ba/F3 cells transfected with the human EPO cDNA clone and some inhibit EPO binding and neutalize EPO-dependent growth.
Fisher et al. Blood 82, 197A (1993) used the same monoclonal antibodies as described in D'Andrea, supra to distinguish erythroid progenitor cells having EPO-dependent growth and maturation from those having EPO-independent growth and maturation.
None of the antibodies described in the aforementioned references were reported to activate the EPO receptor or stimulate the growth and/or maturation of erythroid progenitor cells.
Therefore, it is an object of the invention to produce antibodies which recognize an EPO receptor and bind to it such that the receptor is activated. It is a further object of the invention to produce antibodies which bind to an EPO receptor and stimulate erythropoiesis by stimulating the proliferation and/or differentiation of erythroid progenitor cells to erythrocytes. Such antibodies are useful in the treatment of anemia or in the diagnosis of diseases characterized by dysfunctional EPO receptor. Further, such antibodies may lead to the identification of therapeutic agents for the treatment of anemia.